Phenotypic Mutation 'gandalf' (pdf version)
Allelegandalf
Mutation Type nonsense
Chromosome3
Coordinate20,066,960 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Hps3
Gene Name HPS3, biogenesis of lysosomal organelles complex 2 subunit 1
Synonym(s) Hermansky-Pudlak syndrome 3
Chromosomal Location 20,050,109-20,089,478 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a protein containing a potential clathrin-binding motif, consensus dileucine signals, and tyrosine-based sorting signals for targeting to vesicles of lysosomal lineage. The encoded protein may play a role in organelle biogenesis associated with melanosomes, platelet dense granules, and lysosomes. Mutations in this gene are associated with Hermansky-Pudlak syndrome type 3. [provided by RefSeq, Apr 2015]
PHENOTYPE: Homozygotes for spontaneous null mutations exhibit hypopigmentation and prolonged bleeding associated with a platelet defect. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_080634; MGI: 2153839

MappedYes 
Amino Acid Change Cysteine changed to Stop codon
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000012580] [ENSMUSP00000103957]
AlphaFold Q91VB4
SMART Domains Protein: ENSMUSP00000012580
Gene: ENSMUSG00000027615
AA Change: C667*

DomainStartEndE-ValueType
Pfam:HPS3_N 3 212 2.8e-74 PFAM
Pfam:HPS3_Mid 255 640 1.3e-167 PFAM
Pfam:HPS3_C 649 1000 1.8e-175 PFAM
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000103957
Gene: ENSMUSG00000027615
AA Change: C535*

DomainStartEndE-ValueType
Pfam:HPS3_N 3 87 5.6e-25 PFAM
Pfam:HPS3_Mid 121 508 4.2e-161 PFAM
Pfam:HPS3_C 517 870 9.2e-199 PFAM
Predicted Effect probably null
Meta Mutation Damage Score 0.9755 question?
Is this an essential gene? Probably nonessential (E-score: 0.076) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All alleles(12) : Targeted(4) Gene trapped(1) Spontaneous(6) Chemically induced(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00545:Hps3 APN 3 20073971 missense possibly damaging 0.94
IGL00846:Hps3 APN 3 20079956 missense probably benign 0.00
IGL01320:Hps3 APN 3 20084633 missense probably benign 0.12
IGL01364:Hps3 APN 3 20057469 missense possibly damaging 0.58
IGL01751:Hps3 APN 3 20065130 missense probably damaging 1.00
IGL01843:Hps3 APN 3 20083165 missense probably benign 0.05
IGL02294:Hps3 APN 3 20068212 missense probably damaging 1.00
IGL02581:Hps3 APN 3 20057385 intron probably benign
Blue UTSW 3 20084960 missense probably damaging 1.00
earl_grey UTSW 3 20017173 intron probably benign
pam_gray UTSW 3 20017173 intron probably benign
R0107:Hps3 UTSW 3 20084960 missense probably damaging 1.00
R0245:Hps3 UTSW 3 20066960 nonsense probably null
R0421:Hps3 UTSW 3 20083480 missense probably benign 0.00
R0524:Hps3 UTSW 3 20066940 missense probably damaging 1.00
R0763:Hps3 UTSW 3 20057443 missense probably damaging 1.00
R1795:Hps3 UTSW 3 20066859 critical splice donor site probably null
R1864:Hps3 UTSW 3 20074123 critical splice acceptor site probably null
R2029:Hps3 UTSW 3 20084691 missense probably benign 0.01
R2101:Hps3 UTSW 3 20066947 missense possibly damaging 0.95
R2221:Hps3 UTSW 3 20056527 missense probably benign
R2268:Hps3 UTSW 3 20067099 splice site probably benign
R2520:Hps3 UTSW 3 20083194 missense probably damaging 1.00
R3809:Hps3 UTSW 3 20072976 missense probably damaging 1.00
R3888:Hps3 UTSW 3 20057387 critical splice donor site probably null
R3942:Hps3 UTSW 3 20051103 missense probably damaging 1.00
R4022:Hps3 UTSW 3 20089425 missense possibly damaging 0.69
R4156:Hps3 UTSW 3 20083393 missense probably damaging 1.00
R4739:Hps3 UTSW 3 20084574 critical splice acceptor site probably null
R4823:Hps3 UTSW 3 20066890 missense probably benign 0.03
R4912:Hps3 UTSW 3 20068337 missense probably damaging 1.00
R5307:Hps3 UTSW 3 20066865 missense possibly damaging 0.89
R5859:Hps3 UTSW 3 20063034 missense probably benign 0.02
R6140:Hps3 UTSW 3 20051151 missense probably damaging 1.00
R6183:Hps3 UTSW 3 20063032 missense probably benign 0.04
R6971:Hps3 UTSW 3 20065699 missense probably damaging 1.00
R6981:Hps3 UTSW 3 20076984 missense probably damaging 1.00
R7120:Hps3 UTSW 3 20065705 missense probably damaging 1.00
R7146:Hps3 UTSW 3 20063050 missense probably damaging 1.00
R7223:Hps3 UTSW 3 20084583 missense probably benign 0.05
R7448:Hps3 UTSW 3 20089329 missense probably damaging 0.99
R7452:Hps3 UTSW 3 20065592 missense probably damaging 1.00
R7560:Hps3 UTSW 3 20084616 missense probably benign 0.29
R7659:Hps3 UTSW 3 20076978 nonsense probably null
R7769:Hps3 UTSW 3 20072972 splice site probably null
R8050:Hps3 UTSW 3 20057492 missense probably benign
R8242:Hps3 UTSW 3 20068290 missense possibly damaging 0.59
R8802:Hps3 UTSW 3 20074070 missense probably damaging 1.00
R8822:Hps3 UTSW 3 20057391 missense probably benign
R8945:Hps3 UTSW 3 20068224 missense probably damaging 0.99
R9111:Hps3 UTSW 3 20084575 critical splice acceptor site probably null
R9131:Hps3 UTSW 3 20083350 missense probably damaging 0.98
R9645:Hps3 UTSW 3 20084831 missense probably benign 0.01
R9728:Hps3 UTSW 3 20065128 missense probably benign 0.06
X0021:Hps3 UTSW 3 20084913 missense probably benign 0.14
X0066:Hps3 UTSW 3 20070152 missense probably damaging 1.00
Z1177:Hps3 UTSW 3 20063065 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock Live Mice
MMRRC Submission 038245-MU
Last Updated 2017-05-31 4:22 PM by Katherine Timer
Record Created 2013-05-23 8:58 AM by Tiana Purrington
Record Posted 2014-03-28
Phenotypic Description
Figure 1. The gandalf mouse has gray fur.

Gandalf was initially identified among N-ethyl-N-nitrosourea (ENU)-induced G3 animals as a hypopigmentation mutant (Figure 1). The gandalf homozygous mice have a gray coat. 

Nature of Mutation

Whole exome HiSeq sequencing of the G1 grandsire identified 70 mutations, one of which affected Hps3, a gene known to affect coat pigmentation. Gandalf phenocopies the pam gray phenotype attributed to Hps3. Therefore, the gandalf phenotype was ascribed to the Hps3 mutation, a T to A transversion at base pair 20012796 (v38) on Chromosome 3, or 22515 in the GenBank genomic region NC_000069 encoding Hps3. The mutation corresponds to residue 2046 in the mRNA sequence NM_080634 within exon 11 of 17 total exons.

2029 AGGAAGCTGGATAGTTGTGGAGTTTCACCCGTC
662  -R--K--L--D--S--C--G--V--S--P--V-

The mutated nucleotide is indicated in red.  The mutation results in a cysteine (C) to premature stop codon substitution at residue 667 of the HPS3 protein.

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 2. A, Predicted domains of HPS3. HPS3 contains a putative clathrin-binding (CB) motif, two consensus dileucine-based sorting motifs and an endoplasmic reticulum (ER) membrane retention signal. Amino acid locations are based on the human protein. The location of the gandalf mutation is indicated in red. Image is interactive: click to see other mutations in Hps3 (red indicates phenotypic mutations; green are incidental mutations). B, Components of the biogenesis of lysosomal-related organelle complex 2 (BLOC-2). All three proteins have been shown to co-immunoprecipitate, but only HSP5 and HSP6 bind together in two-hybrid studies suggesting the presence of unknown components of the complex (?).

The gandalf mutation (C667*) may result in nonsense mediated decay of the transcript, or expression of an HPS3 protein truncated after amino acid 667 (Figure 2). The truncated protein would lack one of two dileucine-based sorting motifs and the ER retention signal. Expression and localization of this protein has not been tested. 

Please see the record pam gray for information about Hps3.

Putative Mechanism

Mutations in Hps3 have been documented to result in pigmentation defects [(1); MGI:2153839; pam gray; blue]. Mutations in Hps3 may alter protein (e.g., tyrosinase, Tyrp1, Tyrp2, LAMP1 and LAMP3) trafficking during the maturation of melanosomes, resulting in hypopigmentation (2;3).

Primers PCR Primer
gandalf_pcr_F: TGTTGTGCTTTCAGTGAACACCTACTC
gandalf_pcr_R: AGACAGATCATGTCAGCTCAGTTTTGG

Genotyping
DNA trace file of genotyping. Chr. + shown.

Gandalf genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition.
 

PCR Primers

Gandalf(F): 5’- TGTTGTGCTTTCAGTGAACACCTACTC -3’

Gandalf(R): 5’- AGACAGATCATGTCAGCTCAGTTTTGG -3’

Sequencing Primer

Gandalf_seq(F): 5’- TGGCTCTGGACATAGCTTTATAC -3’
 

PCR program

1) 94°C             2:00

2) 94°C             0:30

3) 55°C             0:30

4) 72°C             1:00

5) repeat steps (2-4) 40X

6) 72°C             10:00

7) 4°C               ∞

The following sequence of 706 nucleotides is amplified (Chr. 3: 20012389-20013094, GRCm38):

  1 tgttgtgctt tcagtgaaca cctactctaa ttgttggctc tggacatagc tttatactct

 61 tctgtgtgaa caaacataat attctgaata tatgtggtac agtgatgatg aaagatcttt

121 cagtagccac tgctgatatt tatcaatctt ttttatttcc atttgacaga tctaaagttc

181 catctaacac tgtctgtttt ctgttactaa gaaaccatga ggtacaaaga agccagaaga

241 aagcccaatg gctcaggaac tactctaatt ctctttcaga cttgttaaca ccaggcattg

301 tactatacct ccgaatggct tttcatctca tttctgtaca tgtcaagatc tcccattttc

361 aaggccactg cagccttggt caaggtcact aagacgggtg aaactccaca actatccagc

421 ttccttaaat aatgcaaggc agttaaggga tcaatattct tcatagaagg gctagagaga

481 acatgaggca gctgcttcgg ctcagccatg tggaatatct gagccacttt tgctgctaat

541 tcctttgatg aatatgaaca aaaaataaac cgttagccaa taggaaacag gggacaaaaa

601 ggaaggaagg tataaaatgc actagataaa taatataatg ctattgagaa agacagagat

661 tacgaaaata tttgaaaggc caaaactgag ctgacatgat ctgtct

Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (A>T, Chr. + strand; T>A, sense strand).

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsTiana Purrington